Deciphering Clonal Evolution of Immunoglobulin Heavy Chain Locus Enhances Next-Generation Sequencing-Based Measurable Residual Disease Detection in Adult B-Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common forms of acute leukemia in adults. Although current therapies have dramatically improved the prognosis of patients with B-ALL, relapse due to measurable residual disease (MRD) remains a major limiting factor in outcome. Malignant cells of B-ALL primarily arise from the Pro-B or Pre-B cell stages, where recombination of variable (V), diversity (D), and joining (J) segment genes could continually occur under the action of RAG recombinase to promote the evolution of immunoglobulin (Ig) genes, especially for Ig heavy chain (IGH). These evolving IGH clones are formed by recombination of incomplete DJ_H rearrangements with multiple V_H fragments or by V_H replacement and are skewed between adult and pediatric B-ALL because of the different genetic backgrounds. Evolving IGH clones could represent a component of MRD, or disappear after treatment. However, the value of evolving IGH clones in sequencing-based MRD monitoring has not been fully investigated.

To detect MRDs in adult B-ALL patients, we established a next-generation sequencing (NGS)-based technology for Ig gene sequencing of IGH (including incomplete IGH), IGK (including Kde), and IGL. The dominant clones used for MRD monitoring were identified in diagnostic samples by their most frequent and discontinuously distributed frequencies. The evolving IGH clones were identified as a group of clones sharing the same 3' of D segment, D-J junction, and J segment. The limit of detection (LOD) reached 1.62 malignant cells per million nucleated cells, with repeatability of 0.999, and linearities of 0.999 and 0.916 in cell lines and clinical samples, respectively.

To address the performance of NGS-based MRD monitoring in adult B-ALL, we intend to test the patients’ specimens at three time points sequentially: at diagnosis, after the first induction chemotherapy, and 3 months after remission for MRD analysis. MRD tests were performed on 67 bone marrow or blood samples from 33 adult B-ALL patients, including 33 samples at diagnosis, 24 samples at the end of induction (EOI, 28 days), and 10 samples at the end of consolidation (EOC, 90 days), respectively, using NGS (NGS-MRD, cutoff 0.0001%), quantitative PCR (qPCR-MRD, cutoff 0.001%, for Ph⁺ ALL), and multiparameter flow cytometry (MFC-MRD, cutoff 0.01%). NGS-, qPCR-, and MFC-MRD detection rates were 79.2% (19 of 24), 60.0% (6 of 10), and 33.3% (8 of 24) at EOI, and 50.0% (5 of 10), 0 % (0 of 5), and 0 % (0 of 9) at EOC. For the 8 MFC-MRD and 6 qPCR-MRD positive tests, the concordance rates with NGS-MRD were 87.5% and 83.3%, respectively. For the 25 MFC-MRD and 9 qPCR-MRD negative tests, the positive rates of NGS-MRD were 64.0% and 77.8%, respectively. This highlighted the advanced sensitivity of NGS-MRD over both qPCR-MRD and MFC-MRD.

In NGS-MRD research of pediatric B-ALL, the clonal clearance of Ig light chains (IGK/IGL) was reported showing no significant difference from the Ig heavy chain (IGH) at both EOI and EOC. However, the value of light chains in NGS-MRD of adult B-ALL was still unknown. To address this question, the clonal clearance of IGH and IGK/IGL was investigated at both EOI and EOC in adult B-ALL. No significant difference was observed at EOI (21.1% vs. 25.0%, p = 1.0000) the same as pediatric B-ALL. However, the clonal clearance of IGK/IGL was significantly less than IGH at EOC (60.0% vs. 100.0%, p = 0.0310), emphasizing the crucial value of light chains monitoring at EOC in NGS-MRD, especially for adult B-ALL.

Finally, to explore the value of evolving IGH clones in NGS-MRD of adult B-ALL patients, clonal evolutions were investigated in 33 diagnostic and 24 paired EOI samples. Evolving IGH clones were obtained in 15 diagnostic and 5 EOI samples, respectively, showing enrichment in the progenitor B stage (p = 0.0390). Additionally, completely rearranged evolving IGH clones were identified in two patients (6%) who harbored dominant clones only from incomplete DJ_H rearrangements, suggesting the use of evolving IGH clones for MRD monitoring could enhance the sensitivity of NGS-MRD detection. In summary, we established a robust NGS-MRD technology for B-ALL. Our findings revealed the necessity of Ig light chain detection and deciphered the value of IGH clonal evolution in NGS-MRD for adult B-ALL.